1. Field of the Invention
This invention relates, generally, to protection against and resistant to pathogen attacks in grapes. More particularly, it relates to novel, isolated proteins found in muscadine grape and Florida hybrid Bunch grape to enhance defense mechanism and tolerance of V. vinifera and Vitis spp. against Xylella fastidiosa, anthracnose, and other pathogenic diseases.
2. Description of the Prior Art
Commercial grape cultivation is dominated by bunch (Vitis vinifera L., Vitis labrusca L., and other Vitis spp.) and Florida hybrid bunch grape (Vitis spp.) genotypes, while muscadine grape (V. rotundifolia) genotypes are underutilized. Muscadine grapes (V. rotundifolia) are among the most important Vitis species cultivated in the southern United States and have potential for expanded markets in wine and juice production. Generally considered an underutilized commodity, muscadine grapes have a characteristic aroma and sweetness that make them acceptable as table wines, but few reports are available on their biochemical/molecular characteristics. Muscadine grape genotypes contain several unique phenolic compounds that would enable them to resist against microbial infection [2, 8, 11]. In addition, muscadine grapes contain higher levels of polyphenols that are known to have beneficial nutraceutical properties. Resveratrol, as well as other grape phenols like catechins, phenolic acids (gallic acid and caffeic acid), caftaric acid, malvidin-3-glucoside, peonidin-3-glucoside, cyanidin-3-glucoside and epigallocatechin-3 gallate have been linked to the reduced cardiac disease and cancer rates observed in wine drinkers. In addition, they have been reported to reduce low-density lipoprotein (LDL)-cholesterol oxidation, a key component of atherosclerosis. It is believed that because of its highly hydrophilic and lipophilic properties, it can provide more effective protection than other well-known antioxidants, such as vitamins C and E.
Resveratrol (3,4,5-trihydroxystilbene) is a phytoalexin, a class of antimicrobial compounds produced as part of plant's defense system against pathogen infection. Resveratrol is considered a biochemical precursor of viniferins and a major stilbene phytoalexin [14]. Stilbene phytoalexin formation is controlled by stilbene synthase, which comprises a small gene family in most species. At the molecular level, no reports are available on muscadine grape. The knowledge of phenolic composition in grape berries and their health benefits is of importance in relation to food quality and development of value added products.
Phytoalexins are known to be important natural components in the defense of plants against pathogen infection. Since the first report of increased disease resistance in transgenic tobacco plants based on an additional foreign phytoalexin [Stilbene Synthase (STS) from V. vinifera] appeared in Nature [7], it has diversified the ways to tackle plant diseases. STS derived from V. vinifera was found to be less effective against fungal diseases [14, 1]. Therefore, an embodiment of the current invention isolates and characterizes muscadine stilbene synthase gene, as muscadine grape berries have higher levels of potent polyphenols than the other Vitis species.
Stilbene synthase gene was isolated and characterized because resveratrol is present in trans-form in muscadine, which is the most active form. Previous studies have shown significant differences in the phenolics content as well as antioxidant activity among the muscadine genotypes. In addition, seed extracts of muscadine cultivars were found to inhibit colon, lung and breast cancer cells growth by 60% (Basha et al.; Personal communication).
Muscadine grape species are native to southeastern USA [1] and are more tolerant to most diseases including Pierce's disease (PD) than bunch grapes [2]. V. vinifera cultivars are highly susceptible to PD [3] while Florida hybrid bunch grape cultivars, which were developed through hybridization of local grape species with the table wine group, V. vinifera [4], are also tolerant to PD. But, their tolerance level varies compared to muscadine as the hybrids contain V. vinifera, a PD-susceptible species in their parentage. PD is caused by Xylella fastidiosa which is vectored by the glassy-winged sharp shooter and thrives in the xylem of grapevines [5]. Through colonization of xylem vessels, X. fastidiosa causes vessel clogging leading to wilting of the plant [6]. Xylem sap is known to contain various amino acids, sugars, organic acids, inorganic ions, proteins, and low concentration of organic compounds essential to support bacterial growth [7, 8].
Amino acids and organic acids are predominant organic compounds in xylem fluid of many woody species [9, 10]. The chemistry of xylem fluid is not fixed and can vary with temperature, time of year, light conditions, water stress, and soil nutrient status [8, 10]. Xylem sap is also known to contain proteins in low concentrations [7, 11, 12] and constitutes an environment in which pathogens can grow leading to vessel clogging and eventual death of the grapevine. Resistance and susceptibility of grapevines to PD could be determined by the interactions occurring within the xylem vessels between the Xylella and xylem sap components.
In both compatible (susceptible) and incompatible (resistance) plant-microbe interactions, plants respond by secreting their own set of proteins [13]. These proteins may either cause direct damage to invaders or play a protective role through inhibition of cell wall-degrading enzymes secreted by pathogens [14] or through oxygen sequestering to maintain the level of oxygen in xylem sap, which may be altered due to pathogen activity. Recent studies have shown that xylem sap proteins of broccoli, rape, pumpkin, cucumber, and tomato share homologies with several pathogen-related proteins including glycine-rich proteins, peroxidase-like proteins, chitinase-like proteins, serine protease-like proteins, aspartyl proteases, and lipid transfer-like proteins which are active in repair and defense reactions of the plant [15]. In tomato, it has been shown that xylem sap protein patterns change in response to infection by pathogenic fungi, and some of the proteins were identified as pathogenesis-related proteins [11, 12]. In addition, appearance of unique proteins has been documented in xylem sap during development of diseases affecting the vascular system [16]. Hence, xylem sap of infected plants may be a rich information source regarding molecular interaction underlying several plant diseases [13, 17]. While the importance of xylem sap proteins in other crops has been established, very little information is available on Vitis xylem sap proteins and their functions.
Anthracnose of grapes (both young leaf and berries) is an economically devastating foliar disease caused by the fungus Elsinoë ampelina Shear. Due to the prevailing hot and humid conditions in Florida, this fungus E. ampelina thrives and devastates grape crop grown under this environment. Symptoms usually appear as numerous circular spots, which enlarge then become sunken and produce lesions with round edges. Once established in a vineyard, the disease can be very destructive. The pathogenic fungus, which attacks all aerial parts of the plants, such as fruits, leaves, tendrils and petioles, is of considerable economic importance [1-3]. The fungus over-winters in dormant and dead canes, making it very difficult to control. Strategies for the control of anthracnose in grapevines, such as developing resistant cultivars are necessary in order to reduce the production cost and environmental impacts of fungicide applications in areas of high disease pressure. For this purpose, the selection of genetic resources showing tolerance to anthracnose is a prerequisite for any breeding program.
It has been reported that, among the grape species, V. vinifera is highly susceptible, while muscadine grapes are mostly tolerant to E. ampelina [6,7]. V. vinifera is one of the finest grapes grown in the world both for table and wine purposes. On the other hand, native muscadine grapes have been considered as one of the most valuable genetic resources in breeding programs for grape disease tolerance [5, 6, 8-13]. As anthracnose is highly prevalent in this part of the world, it is one of the principal factors preventing the development of a grape industry using V. vinifera in the southeastern United States [3]. Growers in this area are forced to grow local species, such as muscadine and Florida hybrid bunch grapes that often compromise the fruit quality. Muscadine grapes have been known for their tolerance or ‘tolerance’ to many diseases found in bunch (Euvitis Planch.) grape species [6,7].
Evaluating and screening of perennial crops, including grapes, for disease tolerance is a constant challenge. Several native grapes and other cultivars (Vitis sp.) have been evaluated for their tolerance to anthracnose [4-7]. This process or approach is time-consuming, laborious and costly. Recently, Yun et al. [12] have developed an efficient and reliable screening process for selecting grape cultivars resistant to anthracnose based on pathogen inoculation and by the application of culture filtrates from E. ampelina, which is accurate, economical and labor-saving.
As of yet, there have been only two formal reports of anthracnose or its causal agent in muscadine grapes [13,14]. Pierce's disease has prevented growing V. vinifera in Floridian and Southeastern United States regions. Muscadine and Florida hybrid bunch grapes (Vitis spp.) can be successfully grown as they are tolerant to Pierce's disease, but their tolerance level to anthracnose varies. The breeding work at the Center for Viticulture and Small Fruit Research, Florida Agricultural and Mechanical University, Tallahassee, Fla., USA has been hampered due to lack of knowledge about the anthracnose tolerance levels in muscadine cultivars used in the breeding program. In a study in 2006 and 2007, 21 (40%) of the 51 muscadine cultivars maintained in the vineyard showed anthracnose symptoms, which were found mainly on young leaves and tendrils as circular or irregular black spots. Hence it was necessary to investigate the level of tolerance of the muscadine cultivars at hand, and to use a more stringent screening process to select muscadine genetic resources that are resistant to anthracnose for use in the ongoing breeding process.
Accordingly, what is needed is more effective mechanisms to be incorporating into the breeding programs of V. vinifera to fighting against and protecting V. vinifera from various pathogenic diseases. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill how the art could be advanced.
While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein.
The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.
In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.